Does anyone know how to add Flag-tag to the C-terminus of the protein? What is the principle of the procedure?
I am planning to transform bacterial gene to mammalian cells and I have to check the transcription of the gene, so I am going to add fag-tag to the C-terminus and then investigate it by monoclonal antibody, but the problem that I have no idea how to do that, can anyone help?
Dear Hadi,
Certain palsmid already gives you the option of C-terminal FLAG tag. You have to clone your insert in-frame of that tag. if you donot have access to such plasmid add codons of FLAG tag followed by a stop codon to 5'-end of your reverse primer. Once again check the frame before ordering primers
Dear Junaid
Thanks for your answer, but I am going to clone my insert into a plasmid which does not give this option
Hadi
If you donot have access to such plasmid add codons of FLAG tag followed by a stop codon to 5'-end of your reverse primer. Once again check the frame before ordering primers
Dear Junaid
In this case the target gene will translate but the flag tag will not translate as a result the protein will not be tagged
thanks
No! see below the C-terminus of translated protein should be like it
XXXXFLAG.
here xxx is your protein followed by FLAF and stop codon (.)
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